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Sadaf M et al. Int. J. Adv. Pharm. Biotech., 2018; 4(1): 23-30
www.ijapbjournal.com IJAPB 23
I J A P B
International Journal of Advances in Pharmacy and Biotechnology Vol.4, Issue-1, 2018, 23-30
ISSN: 2454-8375 Research Article
Open Access
ISOLATION, CHARACTERIZATION, ANALYSIS AND STABILITY STUDY OF SOYA PROTEINS IN SIMULATED FLUIDS
Akshaykumar Bhasme, Tanuja Jadhav, *Sadaf Mutwalli, Swapnil Jadhav
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Near
Chitranagari, Kolhapur -416013.
*Corresponding author e-mail: [email protected]
Received: 6 February 2018 Revised: 26 February 2018 Accepted: 10 March 2018
ABSTRACT: The soyabean is an important dietary component which contains abundant amount of proteins or amino acids required by human body for normal growth and maintenance. In this project, isolation of soya proteins is carried out by extraction with 0.01N sodium bicarbonate and thereafter homogenization, centrifugation and dialysis of extract against Tris buffered saline solution (pH 8.8). These proteins are characterized qualitatively by electrophoresis and quantitatively by Biuret assay method. Biuret assay method effectively quantifies the amount of protein in a given sample. Amino acids present in the isolated protein were identified and quantified using standard or marker amino acids. The stability study of isolated soya proteins is carried out in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), prepared according to protocol specified in USP. These proteins were quantified by Biuret assay method after respective time interval in above specified fluids. The soya proteins are found to hydrolyze in SGF after 5 minutes and in SIF within one hour. Thus in-vitro stability study of proteins in simulated fluids is an effective approach and alternative to animal model for bio-stability assessment. Key words: Stability of soya protein; Simulated Intestinal Fluid; Simulated Gastric Fluid; soya bean
1. INTRODUCTION
Gross chemical composition of food includes
cereals and cereal products, legumes and oil
seeds, fruits and vegetables, meat, fish and
their products, milk and dairy products.
Composition of food constituents includes
proteins, carbohydrates, lipids, enzymes,
water and minerals. Among all the
ingredients present in food, proteins are an
essential nutrient for humans as they play an
important role in the structure and function
of living organisms. Proteins are available
from different food sources such as seafood,
white-meat poultry, milk, cheese, yogurt,
eggs, beans, pork tenderloin, soy, lean beef. [1]
Soya bean is the most commonly consumed
crop in the world as it contains approximately
40% protein, 35% carbohydrate, 20% lipid,
5% ash. Soy protein is a ‘complete protein’ as
it provides all essential amino acids for
human nutrition like lysine and methionine.[2]
United States Food and Drug Administration
approved the claim that 25 g of soy protein in
a day, as a part of diet low in saturated fat and
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cholesterol, may reduce the risk of heart
disease. This was done by different long-term
studies on the effect of soy protein on
cardiovascular diseases. [3] In addition to that
they have also shown antihypertensive
activity, antihyperlipidemic activity,
prevention against osteoporosis, cancer. [3, 4]
Soya protein needs to go through thermal
treatment to inactivate anti-nutritive factors
such as trypsin inhibitor. Jookyeong Lee
optimized the hydrolysis conditions of soy
protein for enhanced solubility, thermal
stability and bioactivity, maintaining low
degree of hydrolysis (DH); in that they
determined the solubility and thermal
stability in solution of hydrolysates and soy
protein isolate at different protein
concentrations. Hydrolysis affects thermal
stability of soya protein at pH 4.5 and below
pH 3.5; soya protein hydrolysates displaced
greater and lower heat stability respectively
in comparison to soya protein isolates. [5]
In this project, stability studies of soya
proteins in simulated gastric and intestinal
fluids were carried out so as to identify their
metabolism profile in stomach and intestine
and thereby compare it with in vivo study
data.
2. MATERIALS AND METHODS
Materials - Sodium bicarbonate (NaHCO3),
Sodium chloride (NaCl) were purchased from
Merck Life Science private limited. TRIS
buffered saline pH 8.8, Pepsin and Pancreatin
were obtained from Himedia, Monobasic
Potassium Phosphate was purchased from
Loba Chemie Pvt. Ltd.
Instruments - UV-Visible Spectrophotometer
(Jasco model V- 530), Electrophoresis
(Biotech R and D laboratories) Centrifuge
(Remi Manufacturer, Model No- C- 851/8)
2.1. Preparation of TRIS buffer saline solution –
Accurately 6.05gm of TRIS and 8.76 gm of
sodium chloride was dissolved in 800 ml of
distilled water. The pH was adjusted with 1M
HCl and the volume was made up to 1 liter
with distilled water. This solution remains
stable for 3 months at 40˚C.
2.2. Extraction process of soyabean
Soyabeans were crushed and extracted using
0.01N NaHCO3. The solution was centrifuged
at 2000 rpm for 5 min. Lipids were extracted
with chloroform and dialysis was carried out
against TRIS buffer saline (pH 8.8) using
membrane space (10000 kg Dalton), in which
the protein was released into buffer solution.
2.3. Quantitative Analysis of protein
Preparation of biuret reagent - Copper
Sulphate (1.50 gm) was dissolved in 250 ml of
water and sodium potassium tartarate (4.5
gm) and potassium iodide (2.5 gm) were
added to it. To this solution, 6 M, 50 ml
sodium hydroxide was added and the volume
were adjusted to 500 ml with water.
Biuret assay – The solutions were prepared
for assay as per quantity, specified in Table
No 1. The volume was made up to 10 ml with
Sadaf M et al. Int. J. Adv. Pharm. Biotech., 2018; 4(1): 23-30
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water. These solutions were analyzed in a UV-
Visible spectrophotometer at 624 nm
wavelength. Using same protocol,
quantitative analysis of soya protein of
unknown concentration may be performed.
Preparation of Buffer solution - Dipotassium
hydrogen phosphate (1.452 gm), sodium
dihydrogen phosphate (7.601 gm) and
sodium chloride (4.8 gm) were dissolved in
sufficient water to produce 1 litre of solution.
Electrophoresis - Sufficient amount of buffer
solution was poured in the compartment.
Whatman filter paper No.1 was cut into strips
having a length of 35 cm and width of 5 cm. A
spot of sample was applied on a strip of filter
paper and marked with pencil. The strips
were dipped in buffer solution in such a way
that both ends of the paper were in contact
with solution. The electrodes were adjoined
to compartments; current was allowed to
flow (3.2 volts/cm) and electrophoresis was
performed for 16 to 18 hours. The strips of
filter paper were dried at 100-110OC for 30
min, spots were observed by spraying
ninhydrin reagent and their distances were
recorded.
Standard amino acid solutions were similarly
run on Whatman filter paper and Rf values
were recorded.
Simulated Intestinal Fluid - Monobasic
potassium phosphate (6.8 gm) was dissolved
in 250 ml of water; 77 ml of 0.2 N Sodium
hydroxide and 500 ml of hydrochloric acid
were added. To this solution, 10 gm of
pancreatin was added and pH was adjusted to
6.8 by adding 0.2 N sodium hydroxide or 0.2
N hydrochloric acid. The volume was adjusted
to 1000 ml with water.
Simulated Gastric Fluid - Sodium chloride (2
gm) and pepsin (3.2 gm) were added to 7 ml
of hydrochloric acid. The pH was adjusted to
1.2 and the volume was made up to 1000 ml.
Stability Study of Soya Protein in SGF and SIF -
The stability study of soya proteins was
carried out in SGF and SIF over a period of
time. Its stability was measured at different
intervals of time (as in minutes), 0 min, 5 min,
10 min, 15 min, 30 min, 60 min. and in hours
at 0 hour, 1 hour, 2 hour, 3 hour, and 4 hour.
The content of soy protein present in
simulated gastric fluid and simulated
intestinal fluid was quantified by using Biuret
test as specified above.
3. RESULTS AND DISCUSSION
Human diet comprises of carbohydrates, fats,
proteins, vitamins, minerals and water. Each
of it has its own significance in our body
hence it becomes necessary to provide it the
required amount. Out of these components,
proteins are important structural and
functional units of human body. Proteins have
diverse varieties of biological actions in
human body. These proteins are important
biochemical catalysts in the human body and
study of these has prime importance in
scientific research.
Sadaf M et al. Int. J. Adv. Pharm. Biotech., 2018; 4(1): 23-30
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These soya proteins are important regulators
in pathogenesis of cancer, osteoporosis and
menopausal disorders in females. The soya
proteins are hydrolyzed in human body in the
gastrointestinal tract and may be absorbed
along the GIT at different rates. These
proteins are hydrolyzed by peptidases and
proteases in GIT. Hence it becomes necessary
to study stability of these proteins in
biological fluids like GI fluids. To carry out
this study, it becomes necessary to use animal
model and hence it becomes necessary to
carry out stability study of these proteins in
in-vitro protocols.
3.1. Extraction of soyabean proteins
All the diet used for Soya proteins have their
iso-electric pH value on the acidic side, hence
these are extracted using sodium bicarbonate
solvent. Thereafter this extract was treated
with chloroform so as to remove fatty acids
and other hydrophobic materials in extract.
Thereafter the aqueous layer was partitioned
using a dialysis membrane of 10000 kD using
TRIS buffer solution. The proteins diffuse
across the dialysis membrane into TRIS
buffer solution. The proteins in TRIS buffer
were thereafter used for further analysis.
Quantitative analysis of soya protein
The soya proteins are quantifiable by Biuret
assay protocol and it has been found effective
for accurate and precise quantitative
estimation of soya proteins.
Regression line equation was used for
determining unknown concentration of soya
proteins and calibration curve of isolated
protein shown in Fig. 1.
Fig. 1: Calibration Curve of Isolated Soya Protein by Biuret Assay
Table 1: Biuret assay method
S.No. BSA conc (mg/ml)
BSA Stock (ml)
Biuret reagent
(ml)
1 0 0 2 2 1 0.1 2 3 2 0.2 2 4 3 0.3 2 5 4 0.4 2 6 5 0.5 2 7 Sample 1 ml - 2
Table 2: Quantitative analysis of soya
protein by Biuret test
S.No. Test Solution
(ml)
Absor-bance
Equation of line for Quantitative Estimation
1. 1 0.053 Absorbance=
slope*concentration + Intercept
Absorbance = 0.064914*Concentration
+ (-0.01687)
2. 2 0.098
3. 3 0.178
4. 4 0.247
5. 5 0.334
6. 6 0.352
Sadaf M et al. Int. J. Adv. Pharm. Biotech., 2018; 4(1): 23-30
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From literature survey, it has been found that
following amino acids are present in soya
proteins. Hence using standard amino acids
as markers, the distance travelled by each
amino acid was determined and sample
solution of soya proteins was also processed
for electrophoresis by same method. Thus it
has been found that extracted soya proteins
contain same amino acids as reported in
literature. Arginine, Histidine, Lysine,
Tyrosine, Tryptophan, Phenylalanine, Cystine,
Methionine, Threonine, Leucine, Isoleucine,
Valine, Glutamic acid, Aspartic acid
After literature review, it has been found that
most of in-vitro assay protocols have made
use of SGF and SIF, official in U.S.P. and
results of these in-vitro assays matches with
in-vivo studies. Hence in this protocol, we
have made use of SGF and SIF, official in U.S.P.
The stability study of soya proteins was
carried out in SGF and SIF over a period of
time specified in following table.
Table 3: Stability study of soya protein in SGF and SIF
Stability in SGF and SIF Stability in SGF and SIF
Time (min) Percent
concentration of soya protein
Time (hour) Percent
concentration of soya protein
0 100 0 100
5 91 1 96
10 72 2 67
15 59 3 61
30 43 4 63
60 38
Sadaf M et al. Int. J. Adv. Pharm. Biotech., 2018; 4(1): 23-30
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SGF (Zero hour to One hour) SIF (One hour to 5 hour)
Fig. 2: Overlain Spectrum of Soya Protein Stability Studies
It has been observed that soya proteins are
stable in SGF up to 5 minutes and thereafter
these will be hydrolyzed and hence found
unstable as indicated by their decreasing
concentration with increase in time in SGF as
well as in overlain spectra of soya proteins
concentration over the period of time. The
concentration of proteins is determined by
Biuret assay protocol specified above.
Similarly soya proteins were stable in SIF up
to 1 hour and unstable thereafter as indicated
in table no. 3 as well as in overlain spectra of
soya proteins concentration over the period
of time in the Fig. 2
4. CONCLUSION
Soyabean is an important dietary component
which contains abundant amount of proteins
or amino acids required by human body for
normal growth and maintenance. In this
project, isolation of soya proteins was carried
out by extraction with 0.01 N sodium
bicarbonate and thereafter homogenization,
centrifugation and dialysis of extract against
Tris buffered saline solution. These proteins
were characterized qualitatively by
electrophoresis and quantitatively by Biuret
assay method. Biuret assay method is an
effective method to quantify amount of
protein in given sample. . Amino acids present
in the isolated protein were identified and
quantified using standard or marker amino
acids. The stability study of isolated soya
proteins was carried out in simulated gastric
fluid (SGF) and simulated intestinal fluid
(SIF), prepared according to protocol
specified in USP. These proteins were
quantified by Biuret assay method after
respective time interval in above specified
fluids. The soya proteins were found getting
hydrolyze in SGF after 5 minutes and in SIF
within one hour. Thus in-vitro stability study
of proteins in simulated fluids is an effective
Sadaf M et al. Int. J. Adv. Pharm. Biotech., 2018; 4(1): 23-30
www.ijapbjournal.com IJAPB 29
approach and alternative to animal model for
bio-stability assessment.
ACKNOWLEDGMENT
We thank Dr. H. N. More, Principal, Bharati
Vidyapeeth College of Pharmacy, Kolhapur;
for providing required facilities to carry out
this research work.
ABBREVIATIONS:
SGF Simulated Gastric Fluid; SIF, Simulated
Intestinal Fluid; GIT, Gastro-Intestinal Tract.
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How to cite this article: Sadaf et al., Isolation, characterization, analysis and stability study of soya proteins in simulated fluids. Int. J. Adv. Pharm.
Biotech., 2018; 4(1): 25-30.